Arylsulfonanilide phosphates

ABSTRACT

The invention provides compounds, compositions and methods relating to novel arylsulfonanilide derivatives and their use as pharmacologically active agents. The compositions find particular use as pharmacological agents in the treatment of disease states, particularly cancer, psoriasis, vascular restenosis, infrections, atherosclerosis and hypercholesterolemia, or as lead compounds for the development of such agents.

This application is a continuation of and claims benefit of U.S.application Ser. No. 09/336,062, filed Jun. 18, 1999 now abandoned,which claims the benefit of U.S. Provisional Patent Application No.60/090,681, filed Jun. 25, 1998, the disclosure of each are incoporatedby reference.

FIELD OF THE INVENTION

The present invention relates to arylsulfonanilide phosphates,derivatives and analogs and their use as pharmacologically active agentscapable of lowering plasma cholesterol levels and inhibiting abnormalcell proliferation.

BACKGROUND

Atherosclerosis is a leading cause of death in the United States. Thedisease results from excess cholesterol accumulation in the arterialwalls, which forms plaques that inhibit blood flow and promote clotformation, ultimately causing heart attacks, stroke and claudication. Aprincipal source of these cholesterol deposits is the low-densitylipoprotein (LDL) particles that are present in the blood. There is adirect correlation between LDL concentration and plaque formation in thearteries. LDL concentration is itself largely regulated by the supply ofactive LDL cell surface receptors, which bind LDL particles andtranslocate them from the blood into the cell's interior. Accordingly,the upregulation of LDL receptor expression provides an importanttherapeutic target.

Lipoprotein disorders have been previously called thehyperlipoproteinemias and defined as the elevation of a lipoproteinlevel above normal. The hyperlipoproteinernias result in elevations ofcholesterol, triglycerides or both, and are clinically important becauseof their contribution to atherosclerotic diseases and pancreatitis.

Lipoproteins are spherical macromolecular complexes of lipid andprotein. The lipid constituents of lipoproteins are esterified andunesterified (free) cholesterol, triglycerides, and phospholipids.Lipoproteins transport cholesterol and triglycerides from sites ofabsorption and synthesis to sites of utilization. Cholesteryl esters andtriglycerides are nonpolar and constitute the hydrophobic core oflipoproteins in varying proportions. The lipoprotein surface coatcontains the polar constituents—free cholesterol, phospholipids, and,apolipoproteins—that permit these particles to be miscible in plasma.

Cholesterol is used for the synthesis of bile acids in the liver, themanufacture and repair of cell membranes, and the synthesis of steroidhormones. There are both exogenous and endogenous sources ofcholesterol. The average American consumes about 450 mg of cholesteroleach day and produces an additional 500 to 1,000 mg in the liver andother tissues. Another source is the 500 to 1,000 mg of biliarycholesterol that is secreted into the intestine daily; about 50 percentis reabsorbed (enterohepatic circulation). The rate-limiting enzyme inendogenous cholesterol synthesis is 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase. Triglycerides, which are nonpolar lipidsconsisting of a glycerol backbone and three fatty acids of varyinglength and degrees of saturation, are used for storage in adipose tissueand for energy.

Lipoproteins are classified into groups based upon size, density,electrophoretic mobility, and lipid and protein composition. Very lowdensity lipoproteins (VLDL) are large, triglyceride-rich lipoproteinsthat are synthesized and secreted by hepatocytes. VLDL interacts withlipoprotein lipase in capillary endothelium, and the core triglyceridesare hydrolyzed to provide fatty acids to adipose and muscle tissue.About half of the catabolized VLDL particles are taken up by hepatic LDLreceptors and the other half remain in plasma, becomingintermediate-density lipoprotein (IDL). IDL is enriched in cholesterylesters relative to triglycerides and is gradually converted by hepatictriglyceride lipase to the smaller, denser, cholesterol ester-rich LDL.As IDL is converted to LDL, apolipoprotein E becomes detached, and onlyone apolipoprotein remains, apo B-100.

LDL normally carries about 75 percent of the circulating cholesterol.Cellular LDL uptake is mediated by a glycoprotein receptor molecule thatbinds to apo B100. Approximately 70 percent of LDL is cleared byreceptor uptake, and the remainder is removed by a scavenger cellpathway using nonreceptor mechanisms. The LDL receptors span thethickness of the cell's plasma membrane and are clustered in specializedregions where the cell membrane is indented to form craters calledcoated pits. These pits invaginate to form coated vesicles, where LDL isseparated from the receptor and delivered to a lysosome so thatdigestive enzymes can expose the cholesteryl ester and cleave the esterbond to form free cholesterol. The receptor is recycled to the cellsurface.

As free cholesterol liberated from LDL accumulates within cells, thereare three important metabolic consequences. First, there is a decreasein, the synthesis of HMG-CoA reductase, the enzyme that controls therate of de novo cholesterol biosynthesis by the cell. Second, there isactivation of the enzyme acyl cholesterol acyltransferase (ACAT), whichesterifies free cholesterol into cholesterol ester, the cell's storageform of cholesterol. Third, accumulation of cholesterol suppresses thecell's synthesis of new LDL receptors. This feedback mechanism reducesthe cell's uptake of LDL from the circulation.

Lipoproteins play a central role in atherosclerosis. This associationwith the most common cause of death in the developed world defines theprincipal clinical importance of the hyperlipoproteinemias. Individualswith an elevated cholesterol level are at higher risk foratherosclerosis. Multiple lines of evidence, including epidemiological,autopsy, animal studies and clinical trials, have established that LDLis atherosclerogenic and that the higher the LDL level, the greater therisk of atherosclerosis and its clinical manifestations. A certaindegree of LDL elevation appears to be a necessary factor in thedevelopment of atherosclerosis, although the process is modified by manyother factors (e.g., blood pressure, tobacco use, blood glucose level,antioxidant level, and clotting factors). Acute pancreatitis is anothermajor clinical manifestation of dyslipoproteinemia. It is associatedwith chylomicronemia and elevated VLDL levels. Most patients with acutepancreatitis have triglyceride levels above 2,000 mg/dL, but a 1983 NIHconsensus development conference recommended that prophylactic treatmentof hypertriglyceridemia should begin when fasting levels exceed 500mg/dL. The mechanism by which chylomicronernia and elevated VLDL levelscause pancreatitis is unclear. Pancreatic lipase may act ontriglycerides in pancreatic capillaries, resulting in the formation oftoxic fatty acids that cause inflammation.

Abundant evidence indicates that treatment of hyperlipoproteinemia willdiminish or prevent atherosclerotic complications. In addition to a dietthat maintains a normal body weight and minimizes concentrations oflipids in plasma, therapeutic agents that lower plasma concentrations oflipoproteins, either by diminishing the production of lipoproteins or byenhancing the efficiency of their removal from plasma, are clinicallyimportant.

The most promising class of drugs currently available for the treatmentof hyperlipoproteinemia or hypercholesterolemia acts by inhibitingHMG-CoA reductase, the rate-limiting enzyme in endogenous cholesterolsynthesis. Drugs of this class competitively inhibit the activity of theenzyme. Eventually, this inhibition leads to a decrease in theendogenous synthesis of cholesterol and by normal homeostaticmechanisms, plasma cholesterol is taken up by LDL receptors to restorethe intracellular cholesterol balance.

Through both the release of precursors of LDL and receptor-mediated LDLuptake from the serum, liver cells play a critical role in maintainingserum cholesterol homeostasis. In both man and animal models, an inversecorrelation appears to exist between liver LDL receptor expressionlevels and LDL-associated serum cholesterol levels. In general, higherhepatocyte LDL receptor numbers result in lower LDL-associated serumcholesterol levels. Cholesterol released into hepatocytes can be storedas cholesteryl esters, converted into bile acids and released into thebile duct, or it can enter into an oxycholesterol pool. It is thisoxycholesterol pool that is believed to be involved in end productrepression of both the genes of the LDL receptor and enzymes involved inthe cholesterol synthetic pathway.

Transcription of the LDL receptor gene is known to be repressed whencells have an excess supply of cholesterol, probably in the form ofoxycholesterol. A DNA sequence in the LDL receptor promoter region,known as the sterol response element (SRE), appears to confer thissterol end product repression. This element has been extensivelyinvestigated (Brown, Goldstein and Russell, U.S. Pat. No. 4,745,060 and4,935,363). The SRE can be inserted into genes that normally do notrespond to cholesterol, conferring sterol end product repression of thechimeric gene. The exact mechanism of the repression is not understood.Brown and Goldstein have disclosed methods for employing the SRE in ascreen for drugs capable of stimulating cells to synthesize LDLreceptors (U.S. Pat. No. 4,935,363). It would be most desirable if thesynthesis of LDL receptors could be upregulated at the level of geneexpression. The upregulation of LDL receptor synthesis at this leveloffers the promise of resetting the level of serum cholesterol at alower, and clinically more desirable, level. Presently, however, thereare no cholesterol lowering drugs that are known to operate at the levelof gene expression. The present invention describes methods andcompounds that act to inhibit directly or indirectly the repression ofthe LDL receptor gene, resulting in induction of the LDL receptor on thesurface of liver cells, facilitating LDL uptake, bile acid synthesis andsecretion to remove cholesterol metabolites and hence the lowering ofLDL-associated serum cholesterol levels.

A number of human diseases stem from processes of uncontrolled orabnormal cellular proliferation. Most prevalent among these is cancer, ageneric name for a wide range of cellular malignancies characterized byunregulated growth, lack of differentiation, and the ability to invadelocal tissues and metastasize. These neoplastic malignancies affect,with various degrees of prevalence, every tissue and organ in the body.A multitude of therapeutic agents have been developed over the past fewdecades for the treatment of various types of cancer. The most commonlyused types of anticancer agents include: DNA-alkylating agents (e.g.,cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, afolate antagonist, and 5-fluorouracil, a pyrimidine antagonist),microtubule disruptors (e.g., vincristine, vinblastine, paclitaxel), DNAintercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormonetherapy (e.g., tamoxifen, flutamide). The ideal antineoplastic drugwould kill cancer cells selectively, with a wide therapeutic indexrelative to its toxicity towards non-malignant cells. It would alsoretain its efficacy against malignant cells even after prolongedexposure to the drug. Unfortunately, none of the current chemotherapiespossess an ideal profile. Most possess very narrow therapeutic indexes,and in practically every instance cancerous cells exposed to slightlysublethal concentrations of a chemotherapeutic agent will developresistance to such an agent, and quite often cross-resistance to severalother antineoplastic agents.

Psoriasis, a common chronic skin disease characterized by the presenceof dry scales and plaques, is generally thought to be the result ofabnormal cell proliferation. The disease results from hyperproliferationof the epidermis and incomplete differentiation of keratinocytes.Psoriasis often involves the scalp, elbows, knees, back, buttocks,nails, eyebrows, and genital regions, and may range in severity frommild to extremely debilitating, resulting in psoratic arthritis,pustular psoriasis, and exfoliative psoriatic dermatitis. No therapeuticcure exists for psoriasis. Milder cases are often treated with topicalcorticosteroids, but more severe cases may be treated withantiproliferative agents, such as the antimetabolite methotrexate, theDNA synthesis inhibitor hydroxyurea, and the microtubule disruptercolchicine.

Other diseases associated with an abnormally high level of cellularproliferation include restenosis, where vascular smooth muscle cells areinvolved, inflammatory disease states, where endothelial cells,inflammatory cells and glomerular cells are involved, myocardialinfarction, where heart muscle cells are involved, glomerular nephritis,where kidney cells are involved, transplant rejection, where endothelialcells are involved, infectious diseases such as HIV infection andmalaria, where certain immune cells and/or other infected cells areinvolved, and the like. Infectious and parasitic-agents per se (e.g.bacteria, trypanosomes, fungi, etc) are also subject to selectiveproliferative control using the subject compositions and compounds.

Accordingly, it is one object of the present invention to providecompounds which directly or indirectly upregulate LDL receptor'synthesisat the level of gene expression and are useful in the treatment ofhypercholesterolemia or hyperlipoproteinemia.

A further object of the present invention is to provide therapeuticcompositions for treating said conditions.

A further object of the invention is to provide therapeutic compositionsfor treating pancreatitis.

Still further objects are to provide methods for upregulating LDLreceptor synthesis, for lowering serum LDL cholesterol levels, and forpreventing and treating atherosclerosis.

A further object of the present invention is to provide compounds whichdirectly or indirectly are toxic to actively dividing cells and areuseful in the treatment of cancer, viral and bacterial infections,vascular restenosis, inflammatory diseases, autoinmmune diseases, andpsoriasis.

A further object of the present invention is to provide therapeuticcompositions for treating said conditions.

Still further objects are to provide methods for killing activelyproliferating cells, such as cancerous, bacterial, or epithelial cells,and treating all types of cancers, infections, inflammatory, andgenerally proliferative conditions. A further object is to providemethods for treating other medical conditions characterized by thepresence of rapidly proliferating cells, such as psoriasis and otherskin disorders.

Other objects, features and advantages will become apparent to thoseskilled in the art from the following description and claims.

SUMMARY OF THE INVENTION

The invention provides novel arylsulfonanilide phosphate compounds, aswell as methods and compositions relating to novel arylsulfonanilidephosphates and their use as pharmacologically active agents. Thecompounds and compositions find use as pharmacological agents in thetreatment of disease states, particularly hypercholesterolemia,atherosclerosis, cancer, bacterial infections, and psoriasis, or as leadcompounds for the development of such agents. The compounds of theinvention have the formula:

or a pharmaceutically acceptable salt thereof.

In the above formula, the symbol R¹ represents a hydrogen, (C₁-C₆)alkylor (C₁-C₆)heteroalkyl. The symbols R² and R³ are each independentlyhydrogen, halogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, —OR¹¹ or —NR¹¹R¹²,in which the symbols R¹¹ and R¹² each independently represent hydrogen,(C₁-C₈)alkyl or (C₁-C₈)heteroalkyl. Alternatively, R² and R³, whenattached to adjacent carbon atoms, can be linked together to form afused 5-, 6- or 7-membered ring.

The symbols R⁴ and R⁵ each independently represent hydrogen,(C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl,aryl(C₁-C₄)heteroalkyl, heteroaryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)heteroalkyl. Optionally, R⁴ and R⁵ are linked togetherto form a 5-, 6- or 7-membered ring. Additionally, R⁴ can also representa single bond to the phenyl ring bearing the phosphoryl group. When R⁴is a single bond to the phenyl ring, R⁵ is hydrogen, (C₁-C₈)alkyl,(C₁-C₈)heteroalkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl,aryl(C₁-C₄)heteroalkyl, heteroaryl(C₁-C₄)alkyl orheteroaryl(C₁-C₄)heteroalkyl.

The symbol Ar represents a substituted aryl group selected from thegroup of:

in which X¹ and X² are each independently F, Cl or Br.

The methods of the present invention use pharmaceutical compositionscontaining compounds of the foregoing description of the general FormulaI for the treatment of pathology such as cancer, bacterial infections,psoriasis, hypercholesterolemia, atherosclerosis, pancreatitis, andhyperlipoproteinermia. Briefly, the inventions involve administering toa patient an effective formulation of one or more of the subjectcompositions.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- andmulti-radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, is also meant toinclude those derivatives of alkyl defined in more detail below as“cycloalkyl” and “alkylene.” The term “alkylene” by itself or as part ofanother substituent means a divalent radical derived from an alkane, asexemplified by —CH₂CH₂CH₂CH₂—. Typically, an alkyl group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkcyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “alkoxy,” employed alone or in combination with other termsmeans, unless otherwise stated, an alkyl group, as defined above,connected to the remainder of the molecule via an oxygen atom, such as,for example, methoxy, ethoxy, 1-propoxy, 2-propoxy and the higherhomologs and isomers.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Also included in the term“heteroalkyl” are those radicals described in more detail below as“heteroalkylene” and “heterocycloalkyl.” The term “heteroalkylene” byitself or as part of another substituent means a divalent radicalderived from heteroalkyl, as exemplified by —CH₂—CH₂ —S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini. Still further, for alkyleneand heteroalkylene linking groups, as well as all other linking groupsdescribed herein, no specific orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetaahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “fluoroalkyl,” aremeant to include monofluoroalkyl and polyfluoroalkyl.

The term “aryl,” employed alone or in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated,an aromatic substituent which can be a single ring or multiple rings (upto three rings) which are fused together or linked covalently. The ringsmay each contain from zero to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. The aryl groups thatcontain heteroatoms may be referred to as “heteroaryl” and can beattached to the remainder of the molecule through a carbon atom or aheteroatom. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl,3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5quinoxalinyl, 3quinolyl, and 6-quinolyl.Substituents for each of the above noted aryl ring systems are selectedfrom the group of acceptable substituents described below.

The terms “arylalkyl” and “arylheteroaklyl” are meant to include thoseradicals in which an aryl group is attached to an alkyl group (e.g.,benzyl, phenethyl, pyridylmethyl and the like) or a heteroalkyl group(e.g., phenoxymethyl, 2-pyridyloxymethyl, 1-naphthyloxy-3-propyl, andthe like). The arylalkyl and arylheteroalkyl groups will typicallycontain from 1 to 3 aryl moieties attached to the alkyl or heteroalkylportion by a covalent bond or by fusing the ring to, for example, acycloalkyl or heterocycloalkyl group. For arylheteroalkyl groups, aheteroatom can occupy the position at which the group is attached to theremainder of the molecule. For example, the term “arylheteroalkyl” ismeant to include benzyloxy, 2-phenylethoxy, phenethylamine, and thelike.

Each of the above terms (e.g., “alkyl,” “heteroalkyl” and “aryl”) aremeant to include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the allyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, —halogen, —SiR′R″R″, —OC(O)R′, —CO₂R′,—CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′, —NH—C(NH₂)═NH,—NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and—NO₂ a number ranging from zero to (2N+1), where N is the total numberof carbon atoms in such radical. R′, R″ and R′″ each independently referto hydrogen, unsubstituted (C₁-C₈)alkyl and heteroalkyl, unsubstitutedaryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy orthioalkoxy groups, or aryl-(C₁-C₄)alkyl groups. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include 1-pyrrolidinyl and 4-morpholinyl.

Similarly, substituents for the aryl groups are varied and are selectedfrom: —halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′, —NH—C(NH₂)═NH,—NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃,—CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a numberranging from zero to the total number of open valences on the aromaticring system; and where R′ and R″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl,(unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl ring mayoptionally be replaced with a substituent of the formula—T—C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl ring may optionally bereplaced with a substituent of the formula —A—(CH₂)_(r)—B—, wherein Aand B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—,—S(O)₂NR′— or a single bond, and r is an integer of from 1 to 3. One ofthe single bonds of the new ring so formed may optionally be replacedwith a double bond. Alternatively, two of the substituents on adjacentatoms of the aryl ring may optionally be replaced with a substituent ofthe formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)₂NR′— is selected fromhydrogen or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatorn” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge, S. M., et al, “Pharmaceutical Salts”,Journal of Phannaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide a compound of formula I.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to the compounds of thepresent invention when placed in a transdermal patch reservoir with asuitable enzyme.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

General

The compounds described herein are related to compounds provided in PCTpublications WO 97/30677 and WO 98/05315. More particularly, compoundsare now described having an attached phosphate, phosphate salt, orphosphate ester group. These arylsulfonanilide phosphates are lesslipophilic that the corresponding arylsulfonanilide phenols and areexpected to reduce brain concentrations of the phenol when administeredas a bolus intravenously. Without intending to be bound by anyparticular theory, it was believed that the compounds would be readilyhydrolyzed in vivo to provide the phenol as the active species. However,the compounds of the present invention have demonstrated surprisingstability in tell culture media, dosing solution, and mouse plasma, yetprovide a level of efficacy against a tumor model equivalent to theparent phenol (non-phosporylated compound) which appears to be presentin an amount of only about 4-10% (based on the administeredarylsulfonanilide phosphate). Additionally, the arylsulfonanilidephosphates provide a bulk stability, or improved shelf-life, relative tothe parent phenols.

Embodiments of the Invention

The present invention provides novel arylsulfonanilide phosphatederivatives having the formula:

in which the symbol R¹ represents hydrogen, (C₁-C₆)alkyl or(C₁-C₆)heteroalkyl, preferably hydrogen.

The symbols R² and R³ are each independently hydrogen, halogen,(C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, —OR¹¹ or —NR¹¹R¹², in which R¹¹ andR¹² are each independently hydrogen, (C₁-C₈)alkyl or (C₁-C₈)heteroalkyl.Additionally, when R² and R³ are attached to adjacent carbon atoms, theycan be linked together to form a fused 5-, 6- or 7-membered ring. Inpreferred embodiments, R² and R³ occupy positions on the phenyl ringthat are meta and/or para to the sulfonanilide nitrogen. Morepreferably, R² represents hydrogen, (C₁-C₃)alkyl or (C₁-C₃)alkoxy. Inother preferred embodiments, R³ represents hydrogen, (C₁-C₃)alkyl, —OR¹¹or —NR¹¹R¹², wherein R¹¹ and R¹² are each independently hydrogen,(C₁-C₃)alkyl or (C₁-C₃)heteroalkyl.

The symbols R⁴ and R⁵ are each independently hydrogen, (C₁-C₈)alkyl,(C₁-C₈)heteroalkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl,aryl(C₁-C₄)heteroalkyl, heteroaryl(C₁-C₄)alkyl orheteroaryl(C₁-C₄)heteroalkyl. In one group of embodiments, R⁴ and R⁵ areoptionally linked together to form a 5-, 6- or7-membered ring.Alternatively, R⁴ represents a single bond to the phenyl ring bearingthe phosphoryl group and R⁵ is hydrogen, (C₁-C₈)alkyl,(C₁-C₈)heteroalkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl,aryl(C₁-C₄)heteroalkyl, heteroaryl(C₁-C₄)alkyl andheteroary(C₁-C₄)heteroalkyl.

The symbol Ar is a substituted aryl group selected from:

in which and X¹ and X² are each independently selected from F, Cl andBr.

In one group of preferred embodiments, Ar is pentafluorophenyl. Inanother group of preferred embodiments, Ar is 2,3,4,5-tetrafluorophenyl.In yet another group of preferred embodiments, Ar is3,4,5-trimethoxyphenyl. In still another group of preferred embodiments,Ar is 3-methoxy-4,5-methylenedioxyphenyl.

In addition to the generally preferred substituents provided above, anumber of particular formulae are also preferred. One preferred group ofcompounds are represented by the formula:

In this group of embodiments, R¹-R⁵ can be any of the groups describedabove. Preferably, R¹ is hydrogen. R² is preferably hydrogen,(C₁-C₃)alkyl or (C₁-C₃)alkoxy, and R³ is preferably hydrogen,(C₁-C₃)alkyl, —OR¹¹ or —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently hydrogen, (C₁-C₃)alkyl or (C₁-C₃)heteroalkyl.

In another group of preferred embodiments, the compounds have theformula:

In this group of embodiments, as with those immediately above, R¹-R⁵ canbe any of the groups described for formula I. Preferably, R¹ ishydrogen, R² is hydrogen, (C₁-C₃)alkyl or (C₁-C₃)alkoxy, and R³ ishydrogen, (C₁-C₃)alkyl, —OR¹¹ or —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently hydrogen, (C₁-C₃)alkyl or (C₁-C₃)heteroalkyl. In the mostpreferred embodiments, R¹ is hydrogen, R² is hydrogen and R³ is methoxy,methyl, dimethylamino or hydroxy. R⁴ and R⁵ are preferably hydrogen,(C₁-C₃)alkyl or aryl.

In yet another group of preferred embodiments, the compounds have theformula:

In this group of embodiments, the preferred substituents are the same asthose described for formula Ib.

In still another group of preferred embodiments, the compounds have theformula:

In this group of embodiments, the preferred substituents are the same asthose described for formula Ib.

In still another group of preferred embodiments, the compounds have thegeneral formula I in which R² and R³ are combined to form a fused5-member ring. Preferred compounds in this group of embodiments areexemplified by the compounds:

Synthesis

Compounds of the present invention can be prepared using certainintermediates and methods described in WO 97/30677 and WO 98/05315. Inone group of embodiments, arylsulfonamidophenols can be prepared asdescribed, and the phenolic hydroxy group can then be phosphorylatedusing reagents such as diethylphosphorylchloride ordimethylphosphorylchloride. Additional compounds can be prepared viaester exchange or saponification.

Still other phosphorylation procedures useful in preparing the presentcompounds are described in Silverberg, et al., Tetrahedron Lett.37(6):771-774 (1996), Saulnier, et al., Bioorg. Med. Chem. Lett.4:2567-2572 (1994), and U.S. Pat. No. 5,561,122, the disclosures of eachbeing incorporated herein by reference.

The compounds used as initial starting materials in this invention maybe purchased from commercial sources or alternatively are readilysynthesized by standard procedures which are well know to those ofordinary skill in the art.

Some of the compounds of Formula I may exist as stereoisomers, and theinvention includes all active stereoisomeric forms of these compounds.In the case of optically active isomers, such compounds may be obtainedfrom corresponding optically active precursors using the proceduresdescribed above or by resolving racemic mixtures. The resolution may becarried out using various techniques such as chromatography, repeatedrecrystallization of derived asymmetric salts, or derivatization, whichtechniques are well known to those of ordinary skill in the art.

The compounds of the invention may be labeled in a variety of ways. Forexample, the compounds may contain radioactive isotopes such as, forexample, ³H (tritium) and ¹⁴C (carbon-14). Similarly, the compounds maybe advantageously joined, covalently or noncovalently, directly orthrough a linker molecule, to a wide variety of other compounds, whichmay provide pro-drugs or function as carriers, labels, adjuvents,coactivators, stabilizers, etc. Such labeled and joined compounds arecontemplated within the present invention.

Analysis of Compounds

Representative compounds and compositions were demonstrated to havepharmacological activity in in vitro and in vivo assays, e.g., they arecapable of specifically modulating a cellular physiology to reduce anassociated pathology or provide or enhance a prophylaxis.

Certain preferred compounds and compositions are capable of specificallyregulating LDL receptor gene expression. Compounds may be evaluated invitro for their ability to increase LDL receptor expression usingwestem-blot analysis, for example, as described in Tam et al. (J. Biol.Chem. 1991, 266, 16764). Established animal models to evaluatehypocholesterolemic effects of compounds are known in the art. Forexample, compounds disclosed herein are shown to lower cholesterollevels in hamsters fed a high-cholesterol diet, using a protocol similarto that described in Spady et al. (J. Clin. Invest. 1988, 81, 300),Evans et al. (J. Lipid Res. 1994, 35, 1634), and Lin et al (J. Med.Chem. 1995, 38, 277).

Certain preferred compounds and compositions display specific toxicityto various types of cells. Certain compounds and compositions of thepresent invention exert their cytotoxic effects by interacting withcellular tubulin. For certain preferred compounds and compositions ofthe present invention, that interaction is covalent and irreversible.Compounds and compositions may be evaluated in vitro for their abilityto inhibit cell growth, for example, as described in Ahmed et al. (J.Immunol. Methods 1994, 170, 211). Established animal models to evaluateantiproliferative effects of compounds are known in the art. Forexample, compounds can be evaluated for their ability to inhibit thegrowth of human tumors grafted into immunodeficient mice usingmethodology similar to that described by Rygaard and Povlsen (ActaPathol. Microbiol. Scand. 1969, 77, 758) and Giovanella and Fogh (Adv.Cancer Res. 1985, 44, 69).

Formulation and Administration of Compounds and PharmaceuticalCompositions

The invention provides methods of using the subject compounds andcompositions to treat disease or provide medicinal prophylaxis, toupregulate LDL receptor gene expression in a cell, to reduce bloodcholesterol concentration in a host, to slow down and/or reduce thegrowth of tumors, etc. These methods generally involve contacting thecell with or administering to the host an effective amount of thesubject compounds or pharmaceutically acceptable compositions.

The compositions and compounds of the invention and the pharmaceuticallyacceptable salts thereof can be administered in any effective way suchas via oral, parenteral or topical routes. Generally, the compounds areadministered in dosages ranging from about 2 mg up to about 2,000 mg perday, although variations will necessarily occur depending on the diseasetarget, the patient, and the route of administration. Preferred dosagesare administered orally in the range of about 0.05 mg/kg to about 20mg/kg, more preferably in the range of about 0.05 mg/kg to about 2mg/kg, most preferably in the range of about 0.05 mg/kg to about 0.2 mgper kg of body weight per day.

In one embodiment, the invention provides the subject compounds combinedwith a pharmaceutically acceptable excipient such as sterile saline orother medium, water, gelatin, an oil, etc. to form pharmaceuticallyacceptable compositions. The compositions and/or compounds may beadministered alone or in combination with any convenient carrier,diluent, etc. and such administration may be provided in single ormultiple dosages. Useful carriers include solid, semi-solid or liquidmedia including water and non-toxic organic solvents.

In another embodiment, the invention provides the subject compounds inthe form of a pro-drug, which can be metabolically converted to thesubject compound by the recipient host. A wide variety of pro-drugformulations are known in the art.

The compositions may be provided in any convenient form includingtablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, suppositories, etc. As such the compositions, inpharmaceutically acceptable dosage units or in bulk, may be incorporatedinto a wide variety of containers. For example, dosage units may beincluded in a variety of containers including capsules, pills, etc.

The compositions may be advantageously combined and/or used incombination with other hypocholesterolemic or antiproliferativetherapeutic or prophylactic agents, different from the subjectcompounds. In many instances, administration in conjunction with thesubject compositions enhances the efficacy of such agents. Examplaryantiproliferative agents include cyclophosphamide, methotrexate,adriamycin, cisplatin, daunomycin, vincristine, vinblastine,vinarelbine, paclitaxel, docetaxel, tamoxifen, flutamide, hydroxyurea,and mixtures thereof. Exemplary hypocholesterolemic and/or hypolipemicagents include: bile acid sequestrants such as quaternary amines (e.g.cholestyramine and colestipol); nicotinic acid and its derivatives;HMG-CoA reductase inhibitors such as mevastatin, pravastatin, andsimvastatin; gemfibrozil and other fibric acids, such as gemfibrozil,clofibrate, fenofibrate, benzafibrate and cipofibrate; probucol;raloxifene and its derivatives; and mixtures thereof.

The compounds and compositions also find use in a variety of in vitroand in vivo assays, including diagnostic assays. For example, variousallotypic LDL receptor gene expression processes may be distinguished insensitivity assays with the subject compounds and compositions, orpanels thereof. In certain assays and in in vivo distribution studies,it is desirable to used labeled versions of the subject compounds andcompositions, e.g. radioligand displacement assays. Accordingly, theinvention provides the subject compounds and compositions comprising adetectable label, which may be spectroscopic (e.g. fluorescent),radioactive, etc.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

¹H-NMR spectra were recorded on a Varian Gemini 400 MHz NMRspectrometer. Significant peaks are tabulated in the order: number ofprotons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet;m, multiplet; br s, broad singlet) and coupling constant(s) in Hertz.Electron Ionization (EI) mass spectra were recorded on a Hewlett Packard5989A mass spectrometer. Mass spectrometry results are reported as theratio of mass over charge, followed by the relative abundance of eachion (in parentheses).

Preparation of Synthetic Intermediates

The majority of the starting materials for the synthesis of the examplesof the present invention are available from commercial sources or areknown compounds described in the published literature. Literaturereferences of general utility to the following examples include:

1) Organic Syntheses, Coll. Vol. VII; 1990, Jeremiah P. Freeman, ed.,John Wiley & Sons, 508-511.

2) Robson, P., Smith, T. A., Stephens, R., Tatlow, J., J. Chem. Soc.,1963, 3692-3703.

3) Synthesis of Fluoroorganic Compounds; 1985, Knunyants, I. andYakobson, G., eds., Springer-Verlag, 190.

The synthesis of a selected group of starting materials is exemplifiedas follows in Examples A-K:

Example A

3,5-Dichloro-2,4,6-trifluorophenylsulfonyl chloride

1,3-Dichloro-2,4,6-trifluorobenzene (5.0 g, 25 mmol) and chlorosulfonicacid (10.0 mL, 150 mmol) were mixed at ambient temperature under anitrogen atmosphere and the reaction was heated at 80° C. for 24 h. Themixture was then allowed to cool to ambient temperature and was pouredonto 12 g of crushed ice. The product was extracted with diethyl ether,dried over MgSO₄, and the solvent was evaporated to produce 4.9 g of thetitle compound, which was used without further purification.

MS(EI): 300 (30, M⁺), 298 (28), 263 (100), 199 (80).

Examples B and C

5-Bromo-2,3,4-trifluorophenylsulfonyl chloride (Example B) and2-Bromo-3,4,5-trifluorophenylsulfonyl chloride (Example C)

The title compounds were obtained as a mixture from1-bromo-2,3,4trifluorobenzene by a method similar to that used inExample A.

Example D

2-Bromo-3,4,5,6-tetrafluorophenylsulfonyl chloride

1-Bromo-2,3,4,5-tetrafluorobenzene (5.0 g, 21.8 mmol) was mixed atambient temperature with 20% fuming sulfuric acid (20 mL). The mixturewas heated at 40° C. for 3 h and at 110° C. for 2 h. The reactionmixture was allowed to cool to ambient temperature and poured onto 12 gof crushed ice. The mixture was acidified dropwise with concentrated HCl(2 mL) until a solid, consisting mostly of2-bromo-3,4,5,6-tetrafluorophenylsulfonic acid was formed. The solid wasfiltered, washed with 12N HCl, and dried under-high vacuum to afford 5.3g of 2-bromo-3,4,5,6-tetrafluorophenylsulfonic acid as a whitehygroscopic solid that was used without further purification. To thesulfonic acid (3.0 g, 9.7 mmol) was then added phosphorous pentachloride(8.0 g, 38.4 mmol) in small portions, at ambient temperature (Caution:exothermic reaction with significant evolution of HCl). The reaction wasallowed to stir for 20 minutes after the final addition of phosphorouspentachloride. The reaction mixture was then poured onto crushed ice andthe white solid that formed was filtered and dried to afford 2.8 g ofthe title compound, which was used without further purification.

MS(EI): 328 (30, M⁺), 293 (70), 229 (30), 148 (100).

Example E

3-Bromo-2,4,5,6-tetrafluorophenylsulfonyl chloride

The title compound was synthesized from1-bromo-2,3,4,6-tetrafluorobenzene by a method similar to that used inExample D.

MS(EI): 328 (20, M⁺), 293 (70), 229 (50), 148 (100).

Example F

1-Bromo-3,4,5,6-tetrafluoro-2-[(3-hydroxy-4-methoxyphenyl)aminosulfonyl]benzene

The title compound was prepared in a manner similar to that described inExample 6 of WO 97/30677, beginning with 3-hydroxy-4-methoxyaniline and2-bromo-3,4,5,6-tetrafluorophenylsulfonyl chloride (Example D, above).

¹H-NMR (CDCl₃): δ 7.28 (br s, 1H), 6.69 (m, 3H), 5.72 (s, 1H), 3.82 (s,3H).

MS(EI): 431 (20), 429 (20), 138 (100).

Anal. calcd. for C₁₃H₈BrF₄NO₄S: C, 36.30; H, 1.87; N, 3.26; S, 7.45.Found: C, 36.20; H, 1.90; N, 3.31; S, 7.39.

Example G

1,3-Dichloro-2,4,6-trifluoro-5-[(3-hydroxy-4-methoxyphenyl)aminosulfonyl]benzene

The title compound was prepared in a manner similar to that described inExample 6 of WO 97/3-677, beginning with 3-hydroxy4-methoxyaniline and3,5-dichloro-2,4,6-trifluorophenylsulfonyl chloride (Example A, above).

¹H-NMR (CDCl₃): δ 6.88 (1H, br s), 6.7-6.8 (3H, m), 5.66 (1H, s), 3.85(3H, s).

MS(EI): 402 (15, M⁺), 401 (20), 138 (100).

Anal. Calcd. for C₁₃H₈Cl₂F₃NO₄S: C, 38.83; H, 2.00; N, 3.48; S, 7.97.Found: C, 38.66; H, 1.97; N, 3.39; S, 7.86.

Example H

1-Bromo-2,3,4-trifluoro-5-[(3-hydroxy-4-methoxyphenyl)aminosulfonyl]benzene.

1-Bromo-2,3,4-trifluoro-5-[(3-hydroxy-4-methoxyphenyl)aminosulfonyl]benzeneand1-Bromo-4,5,6-trifluoro-2-[(3-hydroxy-4-methoxyphenyl)amino-sulfonyl]benzenewere prepared in a manner similar to that described above, beginningwith a mixture of 5-bromo-2,3,4-trifluorophenylsulfonyl chloride(Example B) and 2-bromo-3,4,5-trifluorophenylsulfonyl chloride (ExampleC) and 3-hydroxy-4-methoxyaniline. The two isomeric compounds wereseparated by column chromatography (silica gel; ethyl acetate:hexanes,1:4).

¹H-NMR (CDCl₃): δ 7.79 (1H, m), 6.72-6.62 (4H, m), 5.65 (1H, s), 3.85(3H, s).

Example I

2,3,4,5-Tetrafluoro-1-[(3-hydroxy-4-methoxyphenyl)aminosulfonyl]benzene

The title compound was prepared via catalytic hydrogenation of thecompound prepared in Example F above. Briefly, the starting material wasin methanol and placed in a closed vessel. A catalytic amount of 10%Pd/charcoal was added and the mixture was hydrogenated at 60 psi H₂ for4 h. The resulting mixture was filtered through celite, the solvent wasevaporated and the residue was purified by chromatography (silica;EtOAc/Hexane, 1:4) to yield the title compound.

¹H-NMR (CDCl₃): δ 7.43 (1H, m), 6.80 (1H, br s), 6.73-6.60 (3H, m), 5.67(1H, s), 3.84 (3H, s).

MS(EI): 351 (20, M⁺), 138 (100).

Anal. Calcd. for C₁₃H₉F₄NO₄S: C, 44.45; H, 2.58; N, 3.99; S, 9.13.Found: C, 44.39; H, 2.59; N, 3.94; S, 9.24.

Preparation of other intermediate benzenesulfonamidophenols aredescribed in WO 97/30677 and WO 98/05315. For example,2-hydroxy-1-methoxy-4-pentafluorophenylsulfonaridobenzene (Example 6,page 33); 3-hydroxy-1-pentafluorophenylsulfonamidobenzene (Example 9,page 34): 4-hydroxy-1-pentafluorophenylsulfonamidobenzene (Example 10,page 35); 1,3-dimethoxy-2-hydroxy-5-pentafluoro-phenylsulfonamidobenzene(Example 27, page 45); and3-hydroxy-5-methoxy-1-pentafluorophenylsulfonamidobenzene (Example 28,page 46) are described in each of the cited PCT publications.

Example J

3,4,5-Trimethoxybenzenesulfonyl Chloride

3,4,5-Trimethoxybenzenesulfonyl chloride was synthesized from3,4,5-trimethoxyaniline according to the procedure described in G.Pifferi and R. Monguzzi, Journal of Pharmaceutical Sciences, 1973, 62,1393. In this procedure the aniline was dissolved in concentratedhydrochloric acid and to the resulting mixture was added a solution ofaqueous sodium nitrite at 0° C., the resulting mixture containing thedesired diazonium salt was added at 5° C. to a saturated solution ofsulfur dioxide in glacial acetic acid containing substoichiometricamount of cuprous chloride. The mixture was stirred at ambienttemperature for 3 h, poured into cold water, and the product extractedwith dichloromethane. The solvent was evaporated and the solid residuewas recrystallized from hexanes.

Example K

1-[(3-Hydroxy-4-methoxyphenyl)aminosulfonyl]-3,4,5-trimethoxybenzene

To a solution of 3,4,5-trimethoxybenzenesulfonyl chloride (500 mg, 1.88nmol) in methanol (10 mL) was added 3-hydroxy-4-methoxyaniline (523 mg,3.76 mmol) at ambient temperature. After stirring for 1 h, the reactionmixture was concentrated and the crude residue was purified bychromatography over silica to afford 430 mg (62%) of product as finewhite needles, m.p. 145-146° C.

¹H-NMR (CDCl₃): δ 9.74 (1H, s), 9.15 (1H, s), 6.98 (2H, s), 6.78 (1H, d,J=8.8 Hz), 6.63 (1H, d, J=2.6 Hz), 6.50 (1H, dd, J=8.8, 2.6 Hz), 3.76(6H, s), 3.70 (3H, s), 3.68 (3H, s).

Anal. Calcd. for C,₁₆H₁₉N₁O₇S: C, 52.03; H, 5.18; N, 3.79; S, 8.68.Found: C, 51.87; H, 5.28; N, 3.76; S, 8.77. Each of the phenols above,as well as the related members having different substitution patternscan be phosphorylated using known methods including the proceduredescribed in detail in Example 1.

Example 1

This example illustrates the phosphorylation of the2-hydroxy-1-methoxy-4-(pentafluorophenylsulfonamido)benzene to produce5-(pentafluorophenylsulfonamido)-2-methoxyphenyl phosphate.

5-(Pentafluorophenylsulfonamido)-2-methoxyphenyl phosphate

2-Hydroxy-1-methoxy-4-(pentafluorophenylsulfonamido)benzene, prepared asdescribed in WO 97/30677, (3.0 g, 8.2 mmol) and tetrazole (1.4 g, 19.7mmol) were combined in 70 mL of dry THF and N,N-diisopropyldibenzylphosphoramidite (2.8 mL, 8.2 mmol) was added. The reactionmixture was stirred at room temperature for 4.0 hours. At this point,the reaction mixture was cooled to 0° C. in an ice bath and 14% t-butylperoxide (22 mL, 22.1 mmol) was slowly added. The reaction mixture wasstirred for 0.5 hours, then 75 mL of a 10% NaS₂O₃ solution was added andthe resulting mixture was stirred at room temperature for an additional0.5 hours. THF was removed in vacuo, and the aqueous portion wasextracted with EtOAc. The organic extract was dried over anhydrous MgSO₄and the solvent was removed to provide a crude colorless oil that waspurified by silica gel chromatography (3:7 EtOAc:hexanes as eluant). Theproduct fractions were isolated and solvent was removed in vacuo toyield 3.9 g of a thick clear oil.

¹H NMR (CDCl₃): δ 3.73 (s, 3H); 5.05 (t, J=9.1 Hz, 1H); 5.13 (s, 2H);5.16 (s, 2H); 676 (d, J=11.8 Hz, 1H); 7.00 (d, J=11.8 Hz, 1H); 7.06 (s,1H); 7.31 (s,10H).

ES MS: (M-H)=628.1

The intermediate phosphate diester (3.5 g, 5.6 mmol) was dissolved in 50mL of dry ethanol. This was quickly poured into a flask containing 1.0 gof 10% palladium on carbon. Then 4.5 g (55.6 mmol) of cyclohexadiene wasadded and the reaction was allowed to stir at room temperature under ahydrogen atmosphere overnight. The palladium on carbon was removed byfiltration through a layer of celite, and the solvent was removed invacuo. The crude mixture was purified by reverse-phase. HPLC. Removal ofthe solvents gave 1.13 g of the product phosphate as a white solid.

Example 2 Assessment of Biological Activity

The ability of test compounds to arrest the growth of tumor cells inculture was evaluated using HeLa cells, derived from a human cervicaladenocarcinoma, and obtained from the American Type Culture Collection(ATCC, Rockville, Md.). Cells were grown in culture in the usual way.Test compounds were dosed in triplicate at concentrations ranging from 5nM to 50 μM, and the cellular growth rate was calculated by harvestingthe cells after 72 hours of treatment and measuring their metabolicactivity using an Alamar Blue assay (Biosource International, Camarillo,Calif.). The degree of metabolic activity in the culture is proportionalto the number of living cells. See, Ahmed et al., J. Immunol. Methods1994, 170, 211. The change in growth rate for cells treated with testcompounds was normalized to the growth of untreated cells and a plot ofnormalized cellular growth vs. compound concentration was made. Theconcentration at which 50% growth inhibition (GI50) occurred wasdetermined using a curve fitting program.

The following selected example displays potent cytotoxic activity inthis assay.

Compound GI50 (nM) Example 1 15

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ is a memberselected from the group consisting of hydrogen, (C₁-C₆)alkyl and(C₁-C₆)heteroalkyl; R² and R³ are each independently selected from thegroup consisting of hydrogen, halogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl,—OR¹¹ and —NR¹¹R¹², wherein R¹¹ and R¹² are each independently selectedfrom the group consisting of hydrogen, (C₁-C₈)alkyl and(C₁-C₈)heteroalkyl; or R² and R³, when attached to adjacent carbonatoms, can be linked together to form a fused 5-, 6- or 7-membered ring;R⁴ and R⁵ are each independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl, aryl(C₁-C₄)heteroalkyl, heteroaryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)heteroalkyl, and are optionally linked together to forma 5-, 6- or 7-membered ring; or R⁴ represents a single bond to thephenyl ring bearing the phosphoryl group and R⁵ is selected from thegroup consisting of hydrogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, aryl,heteroaryl, aryl(C₁-C₄)alkyl, aryl(C₁-C₄)heteroalkyl,heteroaryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)heteroalkyl; and Ar is asubstituted aryl group selected from the group consisting of:

 wherein X¹ and X² are each independently selected from the groupconsisting of F, Cl and Br.
 2. A compound of claim 1, wherein Ar isselected from the group consisting of


3. A compound of claim 1, wherein Ar is pentafluorophenyl.
 4. A compoundof claim 1, wherein Ar is 2,3,4,5-tetrafluorophenyl.
 5. A compound ofclaim 1, wherein Ar is 3,4,5-trimethoxyphenyl.
 6. A compound of claim 1,wherein Ar is 3-methoxy-4,5-methylenedioxyphenyl.
 7. A compound of claim1, wherein R¹ is hydrogen.
 8. A compound of claim 1, wherein R² isselected from the group consisting of hydrogen, (C₁-C₃)alkyl and(C₁-C₃)alkoxy.
 9. A compound of claim 1, wherein R³ is selected from thegroup consisting of hydrogen, (C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹², whereinR¹¹ and R¹² are each independently selected from the group consisting ofhydrogen, (C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 10. A compound of claim1, having the formula:


11. A compound of claim 10, wherein R¹ is hydrogen.
 12. A compound ofclaim 10, wherein R² is selected from the group consisting of hydrogen,fluorine, (C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 13. A compound of claim 10,wherein R³ is selected from the group consisting of hydrogen,(C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 14. A compound of claim 1, havingthe formula:


15. A compound of claim 14, wherein R¹ is hydrogen.
 16. A compound ofclaim 14, wherein R² is selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 17. A compound of claim 14, wherein R³is selected from the group consisting of hydrogen, (C₁-C₃)alkyl, —OR¹¹and —NR¹¹R¹², wherein R¹¹ and R¹² are each independently selected fromthe group consisting of hydrogen, (C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.18. A compound of claim 10, wherein R¹ is hydrogen, R² is hydrogen andR³ is selected from the group consisting of methoxy, ethoxy, methyl,dimethylamino and hydroxy.
 19. A compound of claim 1, having theformula:


20. A compound of claim 19, wherein R¹ is hydrogen.
 21. A compound ofclaim 19, wherein R² is selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 22. A compound of claim 19, wherein R³is selected from the group consisting of hydrogen, (C₁-C₃)alkyl, —OR¹¹and —NR¹¹R¹², wherein R¹¹ and R¹² are each independently selected fromthe group consisting of hydrogen, (C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.23. A compound of claim 1, having the formula:


24. A compound of claim 23, wherein R¹ is hydrogen.
 25. A compound ofclaim 23, wherein R² is selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 26. A compound of claim 23, wherein R³is selected from the group consisting of hydrogen, (C₁-C₃)alkyl, —OR¹¹and —NR¹¹R¹², wherein R¹¹ and R¹² are each independently selected fromthe group consisting of hydrogen, (C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.27. A compound of claim 23, wherein R¹ is hydrogen, R² is hydrogen andR³ is selected from the group consisting of methoxy, ethoxy, methyl,dimethylamino and hydroxy.
 28. A compound of claim 1, having theformula:


29. A compound of claim 1, having the formula:


30. A compound of claim 1, having the formula:


31. A compound of claim 1, having the formula:


32. A compound of claim 1, having the formula:


33. A compound of claim 1, having the formula:


34. A compound of claim 1, having the formula:


35. A compound of claim 1, having the formula:


36. A compound of claim 1, having the formula:


37. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and a compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ is a memberselected from the group consisting of hydrogen, (C₁-C₆)alkyl and(C₁-C₆)heteroalkyl; R² and R³ are each independently selected from thegroup consisting of hydrogen, halogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl,—OR¹¹ and —NR¹¹R¹² wherein R¹¹ and R¹² are each independently selectedfrom the group consisting of hydrogen, (C₁-C₈)alkyl and(C₁-C₈)heteroaklyl; or R² and R³, when attached to adjacent carbonatoms, can be linked together to form a fused 5-, 6- or 7-membered ring;R⁴ and R⁵ are each independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, aryl, heteroaryl,aryl(C₁-C₄)alkyl, aryl(C₁-C₄)heteroalkyl, heteroaryl(C₁-C₄)alkyl andheteroaryl(C₁-C₄)heteroalkyl, and are optionally linked together to forma 5-, 6- or 7-membered ring; or R⁴ represents a single bond to thephenyl ring bearing the phosphoryl group and R⁵ is selected from thegroup consisting of hydrogen, (C₁-C₈)alkyl, (C₁-C₈)heteroalkyl, aryl,heteroaryl, aryl(C₁-C₄)alkyl, aryl(C₁-C₄)heteroalkyl,heteroaryl(C₁-C₄)alkyl and heteroaryl(C₁-C₄)heteroalkyl; and Ar is asubstituted aryl group selected from the group consisting of:

 wherein X¹ and X² are each independently selected from the groupconsisting of F, Cl and Br.
 38. A composition of claim 37, wherein Ar isselected from the group consisting of


39. A composition of claim 37, wherein Ar is pentafluorophenyl.
 40. Acomposition of claim 37, wherein Ar is 2,3,4,5-tetrafluorophenyl.
 41. Acomposition of claim 37, wherein Ar is 3,4,5-trimethoxylphenyl.
 42. Acomposition of claim 37, wherein Ar is3-methoxy-4,5-methylenedioxyphenyl.
 43. A composition of claim 37,wherein R¹ is hydrogen.
 44. A composition of claim 37, wherein R² isselected from the group consisting of hydrogen, (C₁-C₃)alkyl and(C₁-C₃)alkoxy.
 45. A composition of claim 37, wherein R³ is selectedfrom the group consisting of hydrogen, (C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹²,wherein R¹¹ and R¹² are each independently selected from the groupconsisting of hydrogen, (C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 46. Acomposition of claim 37, having the formula:


47. A composition of claim 46, wherein R¹ is hydrogen.
 48. A compositionof claim 46, wherein R² is selected from the group consisting ofhydrogen, fluorine, (C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 49. A composition ofclaim 46, wherein R³ is selected from the group consisting of hydrogen,(C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 50. A composition of claim 37,having the formula:


51. A composition of claim 50, wherein R¹ is hydrogen.
 52. A compositionof claim 50, wherein R² is selected from the group consisting ofhydrogen, fluorine, (C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 53. A composition ofclaim 50, wherein R³ is selected from the group consisting of hydrogen,(C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 54. A composition of claim 50,wherein R¹ is hydrogen, R² is hydrogen and R³ is selected from the groupconsisting of methoxy, ethoxy, methyl, dimethylamino and hydroxy.
 55. Acomposition of claim 37, having the formula:


56. A composition of claim 55, wherein R¹ is hydrogen.
 57. A compositionof claim 55, wherein R² is selected from the group consisting ofhydrogen,, (C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 58. A composition of claim55, wherein R³ is selected from the group consisting of hydrogen,(C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 59. A composition of claim 37,having the formula:


60. A composition of claim 59, wherein R¹ is hydrogen.
 61. A compositionof claim 59, wherein R² is selected from the group consisting ofhydrogen, (C₁-C₃)alkyl and (C₁-C₃)alkoxy.
 62. A composition of claim 59,wherein R³ is selected from the group consisting of hydrogen,(C₁-C₃)alkyl, —OR¹¹ and —NR¹¹R¹², wherein R¹¹ and R¹² are eachindependently selected from the group consisting of hydrogen,(C₁-C₃)alkyl and (C₁-C₃)heteroalkyl.
 63. A composition of claim 59,wherein R¹ is hydrogen, R² is hydrogen and R³ is selected from the groupconsisting of methoxy, ethoxy, methyl, dimethylamino and hydroxy.